Heat-shock protein 60 translocates to the surface of apoptotic cells and differentiated megakaryocytes and stimulates phagocytosis

Abstract

Heat-shock protein 60 (Hsp60) is a highly conserved stress protein which has chaperone functions in prokaryotes and mammalian cells. Hsp60 is associated with the mitochondria and the plasma membrane through phosphorylation by protein kinase A, and is incorporated into lipid membranes as a protein-folding chaperone. Its diverse intracellular chaperone functions include the secretion of proteins where it maintains the conformation of precursors and facilitates their translocation through the plasma membrane. We report here that Hsp60 is concentrated in apoptotic membrane blebs and translocates to the surface of cells undergoing apoptosis. Hsp60 is also enriched in platelets derived from terminally differentiated megakaryocytes and expressed at the surface of senescent platelets. Furthermore, the exposure of monocytic U937 cells to Hsp60 enhanced their phagocytic activity. Our results suggests that externalized Hsp60 in apoptotic cells and senescent platelets influences events subsequent to apoptosis, such as the clearance of apoptotic cells by phagocytes.

Fig. 1

BOB78 antibody identifies rh-Hsp60. a, b BOB78 antigen was isolated from platelets by immunoprecipitation, and was revealed to be Hsp60 by sequencing. BOB78 antigen from the lysate of MEG-01-derived platelets was immunoprecipitated with BOB78 IgM monoclonal antibodies, complexed with rabbit anti-mouse antibodies, then captured by protein A Sepharose beads. Captured proteins were separated by SDS-PAGE and stained with Gel-Code. a Stained gel shows that BOB78 antigen was precipitated from the MEG-01 platelets lysate. There is a band specific to the reaction between BOB78 monoclonal antibody and the platelet lysate at 67 kDa (bold arrow, lane 2). This band was sent for sequencing by mass spectrometry (MALDI). Lanes 1, 3 and 4 represent control experiments: lane 1 BOB78 antibody supernatant, beads; lane 2 BOB78 antibody, platelet lysate, beads; lane 3 IgM isotype control, platelet lysate, beads; lane 4 platelet lysate, beads. Bands (broken arrows): a immunoglobulin light chain; b immunoglobulin heavy chain; c other proteins in supernatant. b Western blot probed with BOB78 antibody confirms the specificity of the BOB78 antigen band that was sequenced, as a single band at 67 kDa. The Western blot was performed on duplicate lanes corresponding to the immunoprecipitate gel in a . c The reactivity of BOB78 antibody for rh-Hsp60 protein was compared with that of commercial anti-Hsp60 monoclonal IgG. The lanes were loaded with either rh-Hsp60 (thick lines) or lysate from MEG-01 platelets (interrupted lines). The proteins were separated by SDS-PAGE and Western blotting was performed with BOB78, anti-Hsp60 monoclonal (Hsp60 Ab) and isotype control antibodies. Both the BOB78 antibody and the commercial anti-Hsp60 monoclonal antibody identify rh-Hsp60 and Hsp60 from MEG-01 platelets at 67 kDa. No band of identification is seen with the IgM and IgG isotype controls

Fig. 2

Expression profile of Hsp60 in leucocytes and erythrocytes. a Serum-starved leucocytes and monocytes were stained with BOB78 and IgM isotype control antibodies, with secondary detection by anti-mouse FITC-conjugated antibodies. Surface expression was analysed by flow cytometry. Cells were gated by size (forward scatter, FS) into dying cells (A) and viable cells (V). b Apoptotic leucocytes expressed Hsp60 on the surface. c Single peak of Hsp60 detection in permeabilized leucocytes, indicating the presence of intracellular Hsp60 in apoptotic and viable cells. d Erythrocytes were fixed, permeabilized, and stained with BOB78 and glycophorin A (control positive) antibodies, with secondary detection by anti-mouse FITC-conjugated antibodies. e Intracellular glycophorin was detectable in erythrocytes. f Hsp60 was not detected in erythrocytes

Fig. 3

Hsp60 is expressed on the surface of cells from early to late apoptosis. Human leukaemic THP-1 cells were treated with 3 μM camptothecin for 6 h and analysed by flow cytometry. The cells were costained with BOB78 antibody (detected with anti-mouse FITC) and PI, DiOC6 or annexin V. PtdSer exposure on the cell membrane was detected by staining with PE-conjugated annexin V. a Camptothecin-treated THP-1 cells were separated by size by flow cytometry. Apoptotic cells (green, A) are smaller than viable cells (red, V). b Hsp60 was detected using BOB78 antibody by PE-conjugated anti-mouse secondary antibody. A significant proportion of cells which have lost the mitochondrial transmembrane potential, reflected by reduced DiOC6 green fluorescence, express Hsp60 on the surface. Surface expression of Hsp60 is therefore an event which follows loss of mitochondrial transmembrane potential, a key event in the initiation of apoptosis. c Hsp60 is coexpressed with PtdSer on the surface of cell membranes, indicating that Hsp60 surface exposure occurs early in apoptosis. d PI staining was performed to assess membrane permeability. Apoptotic cells express Hsp60 on the surface. Viable cells [59] exclude PI and do not express Hsp60. Apoptotic cells, early or late, express Hsp60. Hsp60 expression in necrotic cells is low as these cells lose intracellular Hsp60 expression on membrane lysis. e, f Hsp60 localizes predominantly to the membranes and blebs (arrows) of (e) apoptotic THP-1 cells treated with 3 μM camptothecin for 6 h (bar 15 μm) and (f) human hepatoma HUH-7 cells treated with 25 μM monensin for 6 h (bar 25 μm). Untreated live cells do not exhibit these Hsp60-rich blebs. Nuclear staining was effected with TOPRO-3 (blue fluorescence). Naked nuclear material probably results from lysis of late apoptotic or necrotic cells

Fig. 4

Detection of Hsp60 on the surface of differentiating megakaryocytes and senescent platelets. a MEG-01 cells were pooled from several days under standard culture conditions and analysed for Hsp60 expression with FITC-conjugated secondary antibodies and flow cytometry. Viability (V) of the cells was verified through exclusion of PI. b Flow cytometry confirms surface expression of Hsp60 in differentiating MEG-01 cells, but minimal surface expression on viable cells. Differentiating MEG-01 cells which stain weakly for PI exhibit strong expression of Hsp60 on the surface. c In late stages of differentiation and apoptosis, MEG-1 cells have scant cytoplasm. Coincident with strong PI staining of their nuclei, surface expression of Hsp60 is reduced. d, e Senescent platelets produced by standard cultures of MEG-01 stain for FITC-conjugated annexin V, indicating PtdSer exposure on the platelet surfaces. f, g Senescent platelets (which stain for annexin V) express Hsp60 on the surface. Hsp60 surface expression and PtdSer externalization are thus common events in platelet ageing and apoptosis. h Terminal differentiation of human megakaryocytic MEG-01 cells culminates in release of platelets through protrusions in the cell membrane, which resemble apoptotic membrane blebbing (bright-field image). Hsp60 (FITC-conjugated antibody secondary detection) and calreticulin (TRITC) appear to be segregated to different intracellular locations. Hsp60 is distributed to the outer cortex of the cytoplasm and membrane blebs (arrow), whilst calreticulin is limited to the inner cytoplasm. (bar 10 μm)

Fig. 5

Hsp60 enhances phagocytosis by monocytic U937 cells. The proportion of U937 cells that had ingested green fluorescent microspheres was determined by flow cytometric detection in the FL1 channel after incubation with beads that were either pretreated with recombinant Hsp60 (a) or BSA (b), or with untreated beads (c). d Background fluorescence of cells in the absence of beads. e A significant increase in phagocytic activity is detected in U937 cells exposed to beads which have been pre-treated with Hsp60 (p < 0.05). Means with standard deviations are: 16.9 ± 2.7 (beads only), 13.4 ± 1.2 (BSA), 43.6 ± 6.3 (Hsp60). The figure represents data from three separate experiments